Naturally pluripotency occurs in early embryos and may be maintained in vitro in cultured Embryonic Stem (ES) cells harvested from blastocysts. Isolated ES cells can maintain their population by proliferating and self-renewing indefinitely and have the potential to differentiate into every lineage type in the body. Self-renewal allows ES cells in culture to undergo numerous cell cycles, including cell division, without losing pluripotency. Mouse ES cells require co-culture with a feeder layer of cells that provide essential factors. The culture medium typically contains leukemia inhibitory factor (LIF) for mouse ES cells, or fibroblast growth factors (FGFs) for human ES cells, to prevent differentiation. The aminopyrimidine, CHIR99021, is an inhibitor of glycogen synthase kinase 3β (GSK-3β). It enables the self-renewal of embryonic stem cells. Polychronopoulos et al., J Med Chem, 2004, 47: 935-946. Without feeders or cytokines, ES cells undergo spontaneous differentiation and lose their pluripotency.
Nuclear reprogramming, the process used to make induced pluripotent stem (iPS) cells, is the reverse of differentiation, in which differentiated cells revert to pluripotent cells. Induced generation of pluripotent stem cells from adult cells is an artificial manipulation that may not produce cells identical to naturally occurring pluripotent stem cells. However, some aspects of iPS cell generation may parallel the innate genetic processes that occur during embryonic development. Takahashi & Yamanaka, Cell, 2006, 126, 663-676, report a method for the generation of germline-competent induced pluripotent stem cells. See also Okita et al., Nature, 2007, 448, 313-317, 317. Pluripotent stem cells were induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. Huangfu et al., Nat Biotechnol, 2008, 26, 1269-1275, report the induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. However, the efficiency of these methods could be improved.
Several types of integrating viral vectors, including retrovirus and lentivirus have been used in iPS cell generation. Oncogenecity and heterogeneity are major concerns in virally induced human iPS cells because iPS derived offspring sometimes develop tumors. As an alternate approach, the protein products of the reprogramming genes have been delivered directly to cells, without viral DNA. Zhou et al., Cell Stem Cell, 2009, 4(5):381-4, report the use of recombinant proteins containing arginine residues at the C-terminus of each factor to reprogram cells with protein treatments plus the histone deacetylase (HDAC) inhibitor, valproic acid (VPA).
Epigenetic modifications are related to the reprogramming of somatic cells. In somatic cells, reprogramming factors are highly methylated in endogenous loci. However, Imamura et al., BMC Dev Biol, 2006, 6:34, report that these factors are hypomethylated in ES cells and iPS cells indicating that their promoters need to be demethylated in order to be reactivated and thereby reprogrammed to iPS cells. Doi et al., Nat Genet, 2009, 41:1350-1353 report that characterization of CpG methylation can help distinguish the identity of cell types such as fibroblasts, ES cells, and iPS cells.
Small molecules have been attempted to overcome these epigenetic blocks and enhance iPS cell generation. Huangfu et al., Nat Biotechnol, 2008, 26(7):795-7, report that the DNA methyltransferase inhibitor 5′-azacytidine increased the reprogramming efficiency. Shi et al., Cell Stem Cell, 2008, 2:525-528, report that a small-molecule inhibitor of G9a histone methyltransferase, BIX-01294, could enhance the induction of reprogramming in neural stem cells. Shi et al., Cell Stem Cell, 2008, 2(6)3:525-8568-574, report that administration of both BIX01294 and BayK8644, in combination with two factors (Oct4 and Klf4), is able to enhance the reprogramming efficiency of mouse neural progenitors and mouse embryonic fibroblasts. Krawetz & Rancourt, Bioessays, 2009, 31(3):336-43, report that Rho-associate kinase (ROCK) inhibitor, Y-27632, augments human iPS cell induction by enhancing cell survival. It has been reported that inhibitors of Wnt signaling, MEK, FGF, and TGF-β receptors also have effects on the generation and maintenance of ground-level pluripotency of iPS cells.
Shi et al., Cell Stem Cell, 2008, 2(6):525-8, report that BIX01294 and BayK8644, in combination with two factors (Oct4 and Klf4), enhanced the reprogramming efficiency of mouse neural progenitors and mouse embryonic fibroblasts. Upadyay et al., J Mol Biol, 2012, 416(3):319-27, report that an analog of BIX-01294 selectively inhibits a family of histone H3 lysine 9 Jumonji demethylases.
References cited herein are not an admission of prior art.